JP6754932B2 - Methods and kits for detecting stem cells - Google Patents

Description

The present invention relates to methods and kits for detecting stem cells. More specifically, the present invention relates to a method for detecting stem cells by detecting podocalyxin contained in a cell culture supernatant or lysate by a lectin-antibody sandwich method.

One of the challenges of regenerative medicine technology using pluripotent stem cells (hereinafter, also simply referred to as “stem cells”) such as artificial pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) is to obtain stem cells of the desired type. When transplanted into the patient's body after differentiation into cells, how is the risk that stem cells remain undifferentiated and are transplanted into the patient's body together with the differentiated cells to become tumorous or cancerous in the patient's body? Is it to prevent?

As a technique that can be used to evaluate contamination of undifferentiated stem cells that may have tumorigenicity, Patent Document 1 describes stem cell-specific sugar chains and lectins having binding properties to the sugar chains. A method for determining the differentiation state of a cell by detecting it using a method is disclosed. In this method, an undifferentiated sugar chain having a sugar chain structure of "Fucα1-2Galβ1-3GlcNAc" and / or "Fucα1-2Galβ1-3GalNAc" is used by using a recombinant protein (rBC2LCN lectin) of a lectin called BC2LCN lectin. It detects markers. The rBC2LCN lectin is a recombinant protein in which BC2LCN lectin (GenBank Accession No. YP_002232818) corresponding to the N-terminal domain of the BC2L-C protein derived from Gram-negative bacteria (Burkholderia cenocepacia) is expressed in transformed Escherichia coli. It recognizes the sugar chain structure.

Further, Patent Document 2 discloses a method for detecting undifferentiated stem cells remaining after differentiation induction treatment by detecting the above-mentioned sugar chains in the culture supernatant of stem cells using rBC2LCN lectin. Patent Document 2 identifies Podocalixin, which is a complex carbohydrate, as an undifferentiated sugar chain marker having a sugar chain structure of "Fucα1-2Galβ1-3GlcNAc" and / or "Fucα1-2Galβ1-3GalNAc".

International Publication No. 2013/128914 International Publication No. 2013/065302 International Publication No. 2012/147992

J. Am. Soc. Nephrol., 2009, Vol.20, No.8, p.1669-1676. Structure, 2010, Vol.18, No.1, p.59-72. Glycobiology, 2013, Vol.23, No.3, p.322-336. J. Biol. Chem., 2011, Vol.286, No.23, p.20345-20353. Stem Cells, 2007, Vol.25, p.723-730. Cancer Research, 1999, Vol.59, p.4715-4719. Glycobiology, 2011, Vol.21, No.9, p.1125-1130. Scientific Reports, 2014, Vol.4, p.4069.

In Patent Document 2, as a preferred embodiment, an undifferentiated sugar chain marker in a culture supernatant of stem cells is captured by a lectin (rBC2LCN lectin) immobilized on a substrate, and the lectin and the undifferentiated sugar chain marker are captured. A "sandwich method" is disclosed in which an undifferentiated sugar chain marker is detected by forming a complex of the above and reacting the complex with another lectin or antibody labeled.

Patent Document 2 cites four lectins called SRL, CGL2, ABA, and XCL as lectins that can be used in the "lectin-lectin sandwich method" in combination with rBC2LCN lectins.

The present invention relates to a lectin and an antibody in a method for detecting stem cells based on an undifferentiated sugar chain marker (podocalyxin) having a sugar chain structure of "Fucα1-2Galβ1-3GlcNAc" and / or "Fucα1-2Galβ1-3GalNAc". The main purpose is to provide a highly sensitive "lectin-antibody sandwich method" by combination.

In order to solve the above problems, the present invention provides the following [1] to [17].
[1] A method for detecting stem cells by detecting podocalyxin contained in a cell culture supernatant or lysate.
The culture supernatant or lysate and the following (Formula 1):

(R1 represents an OH group or any sugar chain; R2 represents an OH group or any sugar chain, protein, lipid, or other molecule.)
Or the following (Equation 2):

(R1 represents an OH group or any sugar chain; R2 represents an OH group or any sugar chain, protein, lipid, or other molecule.)
A procedure for contacting a lectin having a binding property to a sugar chain represented by (1) and an antibody having a binding property to keratan sulfate to form a complex composed of the lectin, podocalyxin, and the antibody.
A method comprising a procedure for detecting the complex.
[2] The method of [1], wherein the antibody is an antibody having binding property to hyposulfated keratan sulfate. [3] The method of [1] or [2], wherein the antibody comprises Gal-GlcNAc (6S) or a tandem repeat thereof in an epitope.
[4] The method according to any one of [1] to [3], wherein the antibody is an antibody produced by hybridoma R-10G (deposit number: FERM BP-11301) or an antibody that competes with the antibody. [5] The method according to any one of [1] to [4], wherein the cells are cells cultured in a serum-containing medium.
[6] The lectin is
A protein comprising the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence in which one or several amino acids of the amino acid sequence are deleted, substituted, inserted, or added, as described in (Formula 1) or The method according to any one of [1] to [5], which is a protein having a binding property to a sugar chain represented by (Formula 2).
[7] A procedure for contacting the culture supernatant or lysate with the lectin to form a first complex composed of the lectin and the podocalyxin contained in the culture supernatant or lysate.
The method of [1] to [6], which comprises a procedure of contacting the first complex with the antibody to form a second complex composed of the lectin, podocalyxin and the antibody.
[8] The method of [7], wherein the lectin is bound to an insoluble carrier.
[9] The following (Formula 1) contained in the cell culture supernatant or lysate:

(R1 represents an OH group or any sugar chain; R2 represents an OH group or any sugar chain, protein, lipid, or other molecule.)
Or the following (Equation 2):

(R1 represents an OH group or any sugar chain; R2 represents an OH group or any sugar chain, protein, lipid, or other molecule.)
It is a method of detecting stem cells by detecting the sugar chain represented by.
A composite comprising the lectin, the sugar chain, and the antibody by contacting the culture supernatant or lysate with a lectin having a binding property to the sugar chain and an antibody having a binding property to keratan sulfate. The procedure for forming the body and
A method comprising a procedure for detecting the complex.

[10] The method according to any one of [1] to [9], further comprising a procedure for determining the presence / absence or abundance of stem cells contained in the cells based on the presence / absence or detection amount of the complex.
[11] The method according to any one of [1] to [9], further comprising a procedure for determining the differentiation state of the cells based on the presence or absence of the complex or the detected amount.

[12] A method for detecting podocalyxin contained in a cell culture supernatant or lysate, wherein the culture supernatant or lysate and the following (Formula 1):

(R1 represents an OH group or any sugar chain; R2 represents an OH group or any sugar chain, protein, lipid, or other molecule.)
Or the following (Equation 2):

(R1 represents an OH group or any sugar chain; R2 represents an OH group or any sugar chain, protein, lipid, or other molecule.)
A procedure for contacting a lectin having a binding property to a sugar chain represented by (1) and an antibody having a binding property to keratan sulfate to form a complex composed of the lectin, podocalyxin, and the antibody.
A method comprising a procedure for detecting the complex.

[13] A kit for detecting stem cells contained in cells by detecting podocalyxin contained in the cell culture supernatant or lysate.
The following (Equation 1):

(R1 represents an OH group or any sugar chain; R2 represents an OH group or any sugar chain, protein, lipid, or other molecule.)
Or the following (Equation 2):

(R1 represents an OH group or any sugar chain; R2 represents an OH group or any sugar chain, protein, lipid, or other molecule.)
Lectins that have binding properties to sugar chains represented by
A kit containing an antibody having binding property to keratan sulfate.
[14] The kit of [13], wherein the antibody is an antibody having binding property to hyposulfated keratan sulfate.
[15] The kit of [13] or [14], wherein the antibody comprises Gal-GlcNAc (6S) or a tandem repeat thereof in an epitope.
[16] The kit according to any one of [13] to [15], wherein the antibody is an antibody produced by hybridoma R-10G (deposit number: FERM BP-11301) or an antibody that competes with the antibody.
[17] The kit according to any one of [13] to [16], wherein the lectin is bound to an insoluble carrier.

Podocalixin is a type 1 transmembrane glycoprotein that has been identified in epithelial glomerular cells (podocytes) and plays an important role in maintaining glomerular function and morphology, as well as in various cancers. It is known to be related to development (see Non-Patent Document 1). In the present invention, the term "podocalyxin" includes any of the full-length protein of podocalyxin and a partial fragment thereof as long as it has the sugar chain structure of the above (formula 1) and / or (formula 2).

Further, in the present invention, the "sugar chain" means a group of compounds having a structure in which monosaccharides are linked in a chain (straight or dendritic branched chain) by glycosidic bonds. Examples of the monosaccharides constituting the sugar chain include hexoses such as glucose (Glc), galactose (Gal), and mannose; deoxyxose sources such as L-fucose (Fuc); N-acetylglucosamine (GlcNAc), N-acetylgalactosamine ( Hexosemin such as GalNAc); sialic acid such as N-acetylneuraminic acid and N-glycolylneuraminic acid; pentose such as xylose and L-arabinose can be mentioned. The number of monosaccharides constituting the "sugar chain" is not particularly limited, and is about 20,000 to tens of thousands.

In the present invention, the "lectin" refers to a partial structure or an entire structure of a sugar chain bound to a complex sugar such as a glycoprotein, a glycolipid, a proteoglycan, a glycopeptide, a lipopolysaccharide, a peptidoglycan, and a glycoside such as a steroid compound. Means a protein that recognizes and binds.

According to the present invention, in a method for detecting stem cells based on an undifferentiated sugar chain marker (podocalyxin) having a sugar chain structure of "Fucα1-2Galβ1-3GlcNAc" and / or "Fucα1-2Galβ1-3GalNAc", a lectin and an antibody are used. The combination provides a highly sensitive "lectin-antibody sandwich method".

It is a graph which shows the result of having evaluated the method of detecting the stem cell by the lectin-antibody sandwich method which concerns on this invention in comparison with the lectin-lectin sandwich method which concerns on a prior art (Test Example 1). It is a graph which shows the evaluation result by comparing the method of detecting stem cells by the lectin-antibody sandwich method which concerns on this invention with the lectin-antibody sandwich method using various antibodies (Test Example 1). It is a graph which shows the result of having evaluated the detection method of the stem cell by the lectin-antibody sandwich method which concerns on this invention (Test Example 1). It is a graph which shows the result (StemSure hPSC medium Δ) of the detection of the stem cells cultured in different kinds of media by the lectin-antibody sandwich method which concerns on this invention (Test Example 2). It is a graph which shows the result (mTeSR1 medium) of detecting the stem cell culture | cultivated in a different kind of culture medium by the lectin-antibody sandwich method which concerns on this invention (Test Example 2). It is a graph which shows the result (MEF-CM) of detecting the stem cell cultured in the culture medium of a different kind by the lectin-antibody sandwich method which concerns on this invention (Test Example 2). It is a graph which shows the result (TeSR-E8 medium) of detecting the stem cell culture | cultivated in a different kind of culture medium by the lectin-antibody sandwich method which concerns on this invention (Test Example 2). It is a graph which shows the result of having evaluated the detection method of the stem cell by the lectin-antibody sandwich method which concerns on this invention (Test Example 3).

Hereinafter, suitable embodiments for carrying out the present invention will be described. It should be noted that the embodiments described below show an example of typical embodiments of the present invention, and the scope of the present invention is not narrowly interpreted by this.

1. 1. Stem cell detection method The stem cell detection method according to the present invention is contained in the cell culture supernatant or lysate as follows (Formula 1):

(R1 represents an OH group or any sugar chain; R2 represents an OH group or any sugar chain, protein, lipid, or other molecule.)
Or the following (Equation 2):

(R1 represents an OH group or any sugar chain; R2 represents an OH group or any sugar chain, protein, lipid, or other molecule.)
Stem cells are detected by detecting the sugar chain represented by.
Specifically, the culture supernatant or lysate, a lectin having a binding property to the sugar chain, and an antibody having a binding property to keratan sulfate are brought into contact with the lectin, the sugar chain, and the antibody. Stem cells are detected by a procedure for forming a complex containing the above-mentioned complex and a procedure for detecting the complex.

It has been clarified that the sugar chain structures represented by (Formula 1) and (Formula 2) are specifically present on the cell surface of undifferentiated cells and in the culture supernatant and are derived from podocalyxin (Patent Document). See 1 and 2). Therefore, the method for detecting stem cells according to the present invention is, more specifically, a method for detecting stem cells by detecting podocalyxin contained in a cell culture supernatant or lysate, which is the culture supernatant or the above-mentioned method. The lysate, a lectin having a binding property to a sugar chain represented by (Formula 1) or (Formula 2), and an antibody having a binding property to keratin sulfate are brought into contact with each other to obtain the lectin, podocalyxin, and the antibody. It is a method including a procedure for forming a complex composed of, and a procedure for detecting the complex.

[Sugar chain structure]
The above (formula 1) represents the sugar chain structure of "Fucα1-2Galβ1-3GlcNAc". The hydroxyl group at the 4-position of GlcNAc may be replaced with a monosaccharide (preferably fucose) or a branched or unbranched oligosaccharide chain (preferably a sugar chain consisting of 2 to 5 sugars). Further, since the sugar chain structure is a sugar chain that is bound to non-reducing terminals such as glycoproteins, glycolipids and sugars at the 1-position position of GlcNAc as a membrane constituent on the surface of stem cells, it is 1 of GlcNAc. The OH group or the non-reducing end of another sugar, protein, lipid, or other molecule may be attached to the position.

The above (formula 2) represents the sugar chain structure of "Fucα1-2Galβ1-3GalNAc". The hydroxyl group at the 1-position position of GalNAc may be replaced with a monosaccharide (preferably fucose) or a branched or unbranched oligosaccharide chain (preferably a sugar chain consisting of 2 to 5 sugars). Further, since the sugar chain structure is a sugar chain that is bound to non-reducing terminals such as glycoproteins, glycolipids and sugars at the 1-position position of GalNAc as a membrane constituent on the surface of stem cells, it is 1 of GalNAc. A non-reducing end of an OH group or other sugar, protein, lipid, or other molecule may be attached to the position of the position.

[Lectin]
BC2LCN lectin or a variant thereof is preferably used as the lectin having a binding property to the sugar chain represented by (Formula 1) or (Formula 2). The amino acid sequence of BC2LCN lectin is shown in SEQ ID NO: 1. The lectin comprises, in addition to the protein comprising the amino acid sequence set forth in SEQ ID NO: 1, an amino acid sequence in which one or several amino acids of the amino acid sequence have been deleted, substituted, inserted or added. A protein (BC2LCN lectin variant) having a binding property to a sugar chain represented by (Formula 1) or (Formula 2) is also preferably used. Further, in addition to these lectins, any lectin that recognizes a sugar chain represented by (Formula 1) and / or (Formula 2) can be used without particular limitation.

BC2LCN lectin is the N-terminal domain of BC2LC protein derived from Gram-negative bacteria (Burkholderia cenocepacia) (see Non-Patent Document 2). A recombinant protein (rBC2LCN lectin) expressing this BC2LCN lectin in Escherichia coli can be preferably used as the lectin having a binding property to the sugar chain represented by (Formula 1) or (Formula 2). The rBC2LCN lectin can be mass-produced by transforming bacteria. Specifically, the BC2LCN gene encoding the amino acid sequence of SEQ ID NO: 1 is incorporated into an expression vector, introduced into a host cell for expression, and if necessary, the protein is purified to prepare rBC2LCN lectin.

The BC2LCN lectin and the rBC2LCN lectin and their variants do not need to include the full length of the amino acid sequence of SEQ ID NO: 1 as long as they have a bond to the sugar chain represented by (Formula 1) or (Formula 2). Further, one or several amino acids may be deleted, substituted, inserted or added in SEQ ID NO: 1. Here, the number represents a natural number of 20 or less, preferably 10 or less, and more preferably 5 or less.

[antibody]
As the antibody having binding property to keratan sulfate, an IgG antibody produced by hybridoma R-10G (deposit number: FERM BP-11301) is preferably used. An antibody produced by the hybridoma R-10G (hereinafter, also referred to as "R-10G antibody") is described in Patent Document 3. The R-10G antibody is a monoclonal antibody obtained by screening a hybridoma prepared using human iPS cells as an immunogen, using human iPS cell positive and human fetal cancer cell negative as an index. The epitope of the R-10G antibody is low-sulfation keratan sulfate (which does not react with high-sulfation keratan sulfate), and the polypeptide portion of the epitope has been shown to be podocalyxin (). See Non-Patent Document 3). Further, according to Non-Patent Document 3, the epitope of the R-10G antibody includes "Gal-GlcNAc (6S)" or a tandem repeat thereof. The R-10G antibody can be prepared according to the method described in Patent Document 3, but a commercially available antibody (for example, Cosmo Bio Co., Ltd., RIT-M001) may be used.

Keratan sulfate contains a repeating structure of "Gal-GlcNAc (6S)", and Gal in "Gal-GlcNAc (6S)" is a sulfation site. The above-mentioned degree of sulfation means the degree of sulfation of Gal.

As an antibody having binding property to keratan sulfate, in addition to the R-10G antibody, an antibody that competes with the antibody can also be used. An antibody that competes with an R-10G antibody means an antibody that competes with an R-10G antibody in binding to keratan sulfate, that is, an antibody capable of binding to an epitope to which the R-10G antibody binds. Antibodies that compete with the R-10G antibody use a conventionally known competitive binding assay to compete for binding of the R-10G antibody to podocalyxin (ie, the candidate antibody competes with the R-10G antibody for podocalyxin). It can be obtained by confirming that it interferes with the combination with. According to this competitive binding assay, a competitive antibody can be obtained even if a specific epitope of the R-10G antibody has not been determined, but the competitive antibody uses hyposulfated keratan sulfate as an epitope, and in particular Gal. -Preferably an antibody containing GlcNAc (6S) or a tandem repeat thereof in the epitope.

Further, the antibody having binding property to keratan sulfate is particularly limited as long as it is an antibody that recognizes keratan sulfate, preferably hyposulfated keratan sulfate, in addition to the R-10G antibody and an antibody that competes with the antibody. Can be used without.

Here, in the present invention, the term "antibody" also includes "functional fragment of antibody". The “functional fragment of an antibody” means a partial fragment of an antibody having an antigen-binding activity, and includes Fab, F (ab') 2, scFv and the like. In addition, Fab', which is a monovalent fragment of the variable region of the antibody obtained by treating F (ab') 2 under reducing conditions, is also included in the functional fragment of the antibody. However, it is not limited to these molecules as long as it has the ability to bind to an antigen. Functional fragments include not only full-length molecules of antibody proteins treated with suitable enzymes, but also proteins produced in suitable host cells using genetically modified antibody genes.

[Stem cells]
A "stem cell" is generally defined as an undifferentiated cell having "self-renewal ability" capable of proliferating while maintaining an undifferentiated state and "pluripotency" capable of differentiating into all three germ layer lines. The stem cells (pluripotent stem cells) to be detected by the detection method according to the present invention are undifferentiated cells having self-renewal ability and pluripotency, and are at least pluripotent stem cells and multiple stem cells. Includes multipotent stem cells. Examples of stem cells include pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent cells (iPS cells) obtained by introducing a reprogramming factor into somatic cells. Can be mentioned. The pluripotent stem cells include somatic stem cells such as mesenchymal stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells and reproductive stem cells. In the present invention, the term "cell" is used to include both stem cells and differentiated cells (somatic cells).

The cell origin is not particularly limited and may be human, monkey, pig, cow, goat, sheep, mouse, rat or the like.

[Culture supernatant / lysate]
Since it has been clarified that the sugar chain structure represented by (Formula 1) and / or (Formula 2) is specifically present on the cell surface of undifferentiated cells and in the culture supernatant (Patent Documents 1 and 1). 2) In the method for detecting stem cells according to the present invention, the sample used for detecting stem cells may be a lysate of cells or a culture supernatant.

Cell culture supernatants and lysates can be prepared according to conventionally known methods. The lysate can be prepared by physically disrupting the cells or chemically solubilizing the cells. A method of solubilizing cells using a surfactant is preferable from the viewpoints that the protein does not undergo heat denaturation, the protein is not inactivated, the protein recovery rate is good, and the operation is simple.

For example, a cell lysate containing a surfactant is added to cell pellets of 5 × 10 ^{6 to} 5 × 10 ⁷ cells, and the cells are suspended and then reacted on ice for 1 to 10 minutes. Then, it may be centrifuged at 20,000 × g for about 15 minutes, and the obtained supernatant may be used as a cell lysate.

As the cell lysing agent, a buffer solution containing a suitable salt (KCl, NaCl, etc.) or a reducing agent such as DTT, to which a surfactant as a cell lysing agent usually used in this field is added, is used. As the buffer solution, a phosphate buffer solution, a Tris buffer solution, a good buffer solution, a glycine buffer solution, a boric acid buffer solution, etc., which have a buffering action at pH 5.0 to 10.0, preferably around pH 7.0 to 8.0, are preferable. .. The buffer concentration in the buffer solution is usually appropriately selected from the range of 10 to 500 mM, preferably 10 to 100 mM. The salt concentration is usually 100-200 mM. The surfactant may be appropriately selected according to conditions such as the type of cells and the pH and salt concentration of the buffer solution used. Examples thereof include NP-40, poly (oxyethylene) nonylphenyl ether (Wako Pure Chemical Industries, Ltd.), TritonX-100, digitonin and the like. The concentration is usually about 0.01 to 1.0% with respect to the total amount of the buffer solution.

The culture supernatant and lysate may contain serum. According to the method for detecting stem cells according to the present invention, the presence of stem cells can be detected with high sensitivity even when the culture supernatant and lysate of cells cultured in a serum-containing medium are used (test). See Example 1).

As the medium, a medium conventionally used for maintaining undifferentiation of stem cells and inducing differentiation may be used. For example, StemSure (registered trademark) hPSC medium (Wako Pure Chemical Industries, Ltd.), Nutristem (registered trademark) (Biological Industries Ltd), ReproFF (ReproCELL), TeSR ^TM- E8 ^TM (STEMCELL Technologies), Essential 8 ^TM Medium (Life Technologies). ), StemPro (registered trademark) hESC SFM (Life Technologies), mTeSR1 (STEMCELL Technologies) and the like can be used.

The serum is not particularly limited, and for example, calf serum (CS), cow fetal serum (FBS), human-derived serum, cow-derived serum, sheep-derived serum, goat-derived serum, monkey-derived serum, horse-derived serum, and rat-derived serum. , Mouse-derived serum, rabbit-derived serum, hamster-derived serum, guinea pig-derived serum, pig-derived serum, bird (chicken) -derived serum, dog-derived serum, cat-derived serum and the like are used. Serum concentrations in culture supernatants and lysates can vary depending on the concentration of serum added to the medium. The concentration of serum added to the medium in normal maintenance culture or differentiation culture of stem cells is about 0.5 to 20% (v / v).

The culture supernatant and the lysate can be used as they are, diluted, or concentrated in advance with an antibody, lectin, or the like without going through a purification step.

Since the method for detecting stem cells according to the present invention is highly sensitive, the amount of the culture supernatant is about 0.1 to 10 μl, and the picomolar (pM) or nanomolar (nM) level (formula 1) and (formula 2) are used. The represented sugar chain can be detected.

[Lectin-antibody sandwich method]
In the procedure of the method for detecting stem cells according to the present invention, a culture supernatant or a lysate (hereinafter, also referred to as “culture supernatant or the like”) is brought into contact with an antibody having binding property to lectin and keratin sulfate to form a lectin and podocalyxin. A complex formation procedure for forming a complex composed of an antibody and a detection procedure for detecting the complex are included. Hereinafter, a case where rBC2LCN lectin and R-10G antibody are used will be specifically described as an example.

In the complex formation procedure, the rBC2LCN lectin and the R-10G antibody may be brought into contact with the culture supernatant or the like at the same time, but it is more possible to react the R-10G antibody after contacting the culture supernatant or the like with the rBC2LCN lectin. preferable. That is, the complex formation procedure includes the first procedure of bringing the culture supernatant and the like into contact with the lectin to form the first complex composed of the rBC2LCN lectin and the podocalyxin contained in the culture supernatant and the like. It preferably comprises a second procedure of contacting one complex with an R-10G antibody to form a second complex composed of rBC2LCN lectin, podocalyxin and R-10G antibody.

The complex formation procedure may be carried out by a homogeneous method without B / F separation, but more preferably with a heterogeneous method in which B / F separation is carried out using an insoluble carrier.

The amount of the culture supernatant and the like, and the amount (concentration) of the lectin and antibody to react with them are appropriately set according to the cell type, the required measurement sensitivity, the measurement method to be used, the measurement device, and the like.

In the method of performing B / F separation using an insoluble carrier, for example, the rBC2LCN lectin bound to the insoluble carrier, the R-10G antibody not bound to the insoluble carrier, and the culture supernatant or the like are brought into contact with each other to form a complex. It is done by forming. More specifically, in the method of performing B / F separation, a culture supernatant or the like is brought into contact with rBC2LCN lectin bound to an insoluble carrier to obtain a first complex composed of rBC2LCN lectin and podocalyxin. By the first step and the second step of contacting the first complex with a free R-10G antibody to obtain a second complex composed of rBC2LCN lectin, podocalyxin and R-10G antibody. Will be done.

As the insoluble carrier for B / F separation, a substrate such as a slide glass, an ELISA plate (microplate), magnetic beads, a filter, a film, or a membrane, which is used in a usual protein immobilization method, can be used. As the material of the base material, glass, silicon, polycarbonate, polystyrene, polyurethane and the like are usually used.

The method for immobilizing the lectin on the insoluble carrier is not particularly limited, and known methods such as a chemical bond method (a method for immobilizing by covalent bond) and a method for physically adsorbing can be applied. It is also possible to immobilize the lectin on an insoluble carrier using a very strong binding reaction such as the avidin-biotin reaction. In this case, the biotinylated lectin in which biotin is bound to the lectin may be immobilized on a streptavidin plate coated with streptavidin. In addition, the lectin may be immobilized on an insoluble carrier via various linkers commonly used in this field.

In the method of performing B / F separation using an insoluble carrier, after the first step of reacting the culture supernatant or the like with the rBC2LCN lectin immobilized on the insoluble carrier, the first complex and the free R-10G antibody are used. A cleaning procedure for removing unwanted substances from the surface of the solid phase may be included before performing the second step of reacting with. In addition, a cleaning procedure may be included after the second procedure and before performing the detection procedure. By the washing procedure, impurities and unreacted R-10G antibody in the sample can be removed from the surface of the solid phase, and only the second complex can be separated on the surface of the solid phase.

In the method without B / F separation, as a method for separating the complex of rBC2LCN lectin, podocalyxin and R-10G antibody, for example, chromatography method, high performance liquid chromatography method, electrophoresis method, capillary electrophoresis method , Capillary chip electrophoresis, for example, a method using an automatic immunoassay device such as LiBAsys (manufactured by Shimadzu Corporation) can be applied.

The detection procedure can be carried out by detecting a second complex composed of rBC2LCN lectin, podocalyxin and R-10G antibody using a labeling substance. As the labeling substance, for example, a substance that specifically binds to enzymes, radioactive isotopes, fluorescent substances, luminescent substances, DNA, RNA, coenzymes or coenzymes used in ordinary immunoassays, etc. (biotin, Avidin), tags, substances that absorb in the ultraviolet to infrared regions, color-developing fine particles, fluorescent fine particles, metallic fine particles, magnetic substances, substances that have properties as spin labeling agents, etc., are usually used in this field. Examples include labeling substances.

In order to bind the labeling substance to the rBC2LCN lectin and / or the R-10G antibody, preferably the R-10G antibody, for example, the labeling method used in ordinary immunoassays or the like may be appropriately used. In addition, a method of binding the labeling substance to the antibody via one or several amino acids or via a linker with one or several amino acids can also be adopted. Further, since various kits for binding the labeling substance to the protein are commercially available, they may be used for labeling according to the instruction manual attached to the kit.

For example, a method for performing B / F separation using rBC2LCN lectin immobilized on an insoluble carrier and a free R-10G antibody labeled with horseradish peroxidase (HRP) as a labeling substance is roughly as follows.

The culture supernatant or the like is brought into contact with an insoluble carrier on which the rBC2LCN lectin is immobilized, and the reaction is carried out at 4 to 40 ° C. for 3 minutes to 20 hours to allow the first complex of the rBC2LCN lectin and podocalyxin on the solid phase surface. To generate. Next, a solution containing the HRP-labeled R-10G antibody was added onto the surface of the solid phase and reacted at 4 to 40 ° C. for 3 minutes to 16 hours to obtain the first immobilized rBC2LCN lectin-podocalyxin-labeled R-10G antibody. Generate two complexes. Subsequently, a TMB (3,3'5,5'-tetramethylbenzidine) solution having an appropriate concentration is added and reacted for a certain period of time. Then, a reaction terminator such as 1M sulfuric acid is added to terminate the reaction, and the absorbance at 450 nm is measured. From the obtained measured value and the calibration curve obtained by performing the same measurement on a solution of podocalyxin whose concentration is known in advance, podocalyxin in a culture supernatant or the like (or (formula 1) or (formula 2) on podocalyxin). The amount of sugar chain represented by) can be obtained.

Further, for example, using rBC2LCN lectin labeled with Alexa Fluor-488 tetrafluorophenyl ester or the like and R-10G antibody labeled with Alexa Fluor-647 succinimidyl ester or the like, according to a known fluorescence cross-correlation spectroscopy (FCCS). The sugar chain represented by (formula 1) or the above (formula 2) can also be measured.

Further, the detection of the complex of "rBC2LCN lectin-podocalyxin-R-10G antibody" is carried out without using a labeling substance, for example, a method of measuring by utilizing the property derived from the complex, specifically, surface plasmon resonance and the like. It is also possible to carry out by a method such as the homogenia swimnoassay system of.

The lectin-antibody sandwich method according to the present invention is not limited to the method used, and can be easily and quickly measured by applying it to a measurement system using an automatic analyzer. There are no particular restrictions on the combination of reagents, etc. when measuring using the method or automatic analyzer, depending on the environment and model of the automatic analyzer to be applied, or taking other factors into consideration. The best combination of reagents and the like may be selected and used. Furthermore, the lectin-antibody sandwich method according to the present invention can also be applied to Micro-TAS (Micro-Total Analysis Systems: μ-TAS, μ comprehensive analysis system).

[application]
Since the sugar chain structures represented by (Formula 1) and (Formula 2) are specifically present on the cell surface of undifferentiated cells and in the culture supernatant, the detection method according to the present invention can be used to culture cells. By detecting the sugar chain on podocalyxin contained in Qing dynasty, the presence or absence of stem cells present in the cells can be detected. Further, by quantitatively detecting the sugar chain, the abundance of stem cells in the cells can be determined.

For example, a certain amount of culture supernatant of stem cells that are being cultured to maintain an undifferentiated state (maintenance culture) is collected with a micropipette or the like, and according to the detection method according to the present invention, (formula 1) and / / on podocalyxin. Alternatively, the sugar chain represented by (Equation 2) is detected. If the sugar chain is detected, it can be confirmed that the cells maintain the undifferentiated state, which makes it possible to obtain high-quality stem cells without contamination of the differentiated cells. Alternatively, it is also possible to determine how much of the total cells maintain the undifferentiated state (conversely, how much is differentiated) based on the quantitative value of the sugar chain. ..

As a means for collecting a certain amount of the culture supernatant or the like, manual work may be used, but it can also be automatically collected by an automatic culture device or the like.

Further, for example, after inducing differentiation of stem cells, a certain amount of cell culture supernatant or the like is collected with a micropipette or the like, and according to the detection method according to the present invention, (formula 1) and / or (formula 2) on podocalyxin. ) Is detected. If the sugar chain is detected, it can be confirmed that undifferentiated stem cells remain in the cells. Conversely, if sugar chains are no longer detected (at the same level as the background value), it can be confirmed that all cells are differentiated, thereby obtaining differentiated cells that are not likely to be contaminated with undifferentiated cells. Is possible. Alternatively, it is also possible to determine how much of the total cells are differentiated (conversely, how many cells remain undifferentiated) based on the quantitative value of the sugar chain.

When a cell culture supernatant is used for the detection method according to the present invention, it is necessary to use the culture supernatant after a detectable amount of the sugar chain of (Formula 1) or (Formula 2) has been secreted. .. The time required for a detectable amount of sugar chains to appear in the culture solution after the exchange varies depending on the cell type and culture conditions and can be appropriately set, but is, for example, about 18 to 30 hours. Since the medium is usually changed every 24 hours, it is preferable to use a part of the culture supernatant to be discarded at that time.

Here, any method may be used as a method for inducing differentiation of stem cells. For example, a method of culturing stem cells in the presence of retinoic acid to differentiate into nervous system cells, a method of forming epidermal cells based on the surface of NIH3T3 cells whose proliferation has been stopped can be applied.

2. 2. Stem cell detection kit The stem cell detection kit according to the present invention is used in the above-mentioned stem cell detection method, and has the following (Formula 1):

(R1 represents an OH group or any sugar chain; R2 represents an OH group or any sugar chain, protein, lipid, or other molecule.)
Or the following (Equation 2):

(R1 represents an OH group or any sugar chain; R2 represents an OH group or any sugar chain, protein, lipid, or other molecule.)
Includes a lectin having a binding property to a sugar chain represented by, and an antibody having a binding property to keratan sulfate.

Preferred embodiments and specific examples of the configuration of the kit are as described in the above-mentioned method for detecting stem cells. Specifically, the antibody having binding property to keratan sulfate is particularly preferably an antibody having binding property to hyposulfated keratan sulfate. In addition, the antibody preferably contains Gal-GlcNAc (6S) or a tandem repeat thereof in the epitope. Specifically, an antibody produced by hybridoma R-10G (deposit number: FERM BP-11301) or an antibody that competes with the antibody can be used.

In addition, the reagents included in the kit include reagents usually used in this field, such as buffers, reaction promoters, sugars, proteins, salts, stabilizers such as surfactants, preservatives and the like. Those that do not inhibit the stability of coexisting reagents and the like and do not inhibit the reaction of podocalyxin with lectins and antibodies may be included. In addition, the concentration may be appropriately selected from the concentration range usually used in this field. In addition, the kit may include a standard used to create a calibration curve for podocalyxin.

In the stem cell detection kit according to the present invention, the lectin having binding to the sugar chain represented by (Formula 1) or (Formula 2) is preferably bound to an insoluble carrier. The antibody having binding property to keratan sulfate is preferably pre-labeled, but the kit may include a reagent for the user to label the antibody at the time of use.

The terms and concepts in the present invention are based on the meanings of terms commonly used in the art, and the various techniques used to carry out the present invention are excluding those techniques for which the source is specified. Can be easily and reliably carried out by those skilled in the art based on known documents and the like.
In addition, various analyzes were performed by applying the methods described in the analytical instruments or reagents used, the instruction manuals for the kits, catalogs, and the like mutatis mutandis.
The technical documents, patent gazettes and patent application specifications cited in the present specification shall be referred to as the description contents of the present invention.

[Test Example 1: Detection of stem cells by lectin-antibody sandwich method]
In order to evaluate the method for detecting stem cells by the lectin-antibody sandwich method according to the present invention, quantitative measurement of podocalyxin was performed using a diluted solution of the culture supernatant of iPS cells. The measurement was performed by an ELISA detection system using a microplate.

Biotinylated rBC2LCN lectin (0.5 μg equivalent) was added to the wells of a streptavidin plate (Sumitomo Bakelite Co., Ltd.) and immobilized at 37 ° C. for 1 hour. The recombinant lectin was prepared according to the method described in Non-Patent Document 4. After washing the wells with a buffer solution (1% Triton X-100, phosphate buffer solution), the culture supernatant of iPS cells (201B7 strain) cultured in mTeSR1 medium for 24 hours was serially diluted with medium (2% FBS, DMEM). 50 μl of the solution was added to the wells and reacted at 37 ° C. for 1 hour. The iPS cells (201B7 strain) were sold by RIKEN BioResource Center.

After washing the wells with a buffer solution, a peroxidase-labeled R10G antibody (0.1 μg / ml) was added to the wells, and the mixture was reacted at 37 ° C. for 1 hour. After washing the wells with a buffer solution, 100 μl of a substrate solution (TMB, Wako Pure Chemical Industries, Ltd.) was added to the wells, and the reaction was carried out at room temperature for 30 minutes. After stopping the reaction by adding 1M sulfuric acid to the wells, the absorbance (450 nm) of each well was measured. For comparison, instead of R10G antibody, rABA lectin (derived from Agaricus bisporus, see Patent Document 2), anti-SSEA3 antibody, anti-SSEA4 antibody, anti-Tra-1-60 antibody, anti-Tra-1-81 antibody, anti-podocalyxin antibody. Was used at the same concentration.

(1) R10G antibody: hyposulfated keratan antibody (Cosmo Bio Co., Ltd., RIT-M001). Recognize low-sulfation keratan sulfate (low-sulfated keratan sulfate) on podocalyxin.
(2) rABA lectin: The amino acid sequence is shown in SEQ ID NO: 2 (see also RCSB Protein Data Bank Accession No. 1Y2V).
(3) Anti-SSEA3 antibody: Rat monoclonal IgM antibody (Millipore, MAB4303). It recognizes SSEA3 antigen (Stage-specific embryonic antigen 3) expressed on the surface of human EC cells (Tetracarcinoma stem cells), human EG cells (Embryonic germ cells), and human ES cells.
(4) Anti-SSEA4 antibody: Mouse monoclonal IgG antibody (Millipore, MAB4304). It recognizes the SSEA4 antigen (Stage-specific embryonic antigen 4) expressed on the surface of human EC cells, human EG cells, and human ES cells.
(5) Anti-Tra-1-60 antibody: Mouse monoclonal IgM antibody (Millipore, MAB4360). Recognize keratan sulfate on podocalyxin (see Non-Patent Documents 5 and 6).
(6) Anti-Tra-1-81 antibody: Mouse monoclonal IgM antibody (Millipore, MAB4381). Recognize keratan sulfate on podocalyxin (see Non-Patent Documents 5 and 6).
(7) Anti-podocalyxin antibody: Goat polyclonal IgG antibody (R & D System. AF1658). Recognize human podocalyxin.

The results are shown in FIGS. 1 and 2. When rABA lectin was used, the influence of the background was remarkable when a highly diluted solution having a dilution factor of 40 times (in the figure, dilution factor: 0.025) or less was used, and the quantitativeness was lowered (). The coefficient of determination of the linear regression line: 0.6387). It was considered that the cause was an increase in background due to non-specific binding of serum components in the medium to rBC2LCN lectins and rABA lectins.

On the other hand, when the R10G antibody was used, the background did not increase due to the non-specific binding as described above at any dilution ratio, and highly accurate quantitative measurement was possible (linear regression line). The coefficient of determination of: 0.9988).

In addition, anti-SSEA3 antibody and anti-SSEA4 antibody could not be measured due to lack of reactivity with the antigen (see FIG. 2). Even when the anti-Tra-1-60 antibody and the anti-Tra-1-81 antibody that recognize keratan sulfate on podocalyxin were used, they hardly or not bound to the antigen, and the measurement was impossible (Fig. 2). reference). The following were considered as the causes.
The anti-Tra-1-60 antibody and the anti-Tra-1-81 antibody are both antibodies using keratan sulfate on podocalyxin as an epitope, but are said to contain "Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc" as an epitope. (See Non-Patent Document 7), the epitope is different from that of the R-10G antibody, which is said to contain "Gal-GlcNAc (6S)" or its tandem repeat in the epitope.
The anti-Tra-1-60 antibody and the anti-Tra-1-81 antibody are said to have hypersulfated keratan sulfate as an epitope, and have different epitopes from the R-10G antibody having hyposulfated keratan sulfate as an epitope.
The anti-Tra-1-60 antibody and the anti-Tra-1-81 antibody are IgM antibodies, unlike the R-10G antibody, which is an IgG antibody.

Furthermore, measurement was not possible when an anti-podocalyxin antibody (R & D System. AF1658) was used. The reason for this is that the antibody was prepared using recombinant podocalyxin expressed in myeloma cells as an antigen, and therefore did not have sufficient reactivity with podocalyxin expressed in stem cells. Conceivable. It is possible that the specific structure of podocalyxin (eg, the sugar chain structure of (formula 1) and (formula 2)) derived from its expression in stem cells inhibited the binding of the antibody to the epitope.

FIG. 3 shows quantitative measurement of podocalyxin using a diluted culture supernatant solution in the same manner as above except that the serum concentration of iPS cells during culture was changed from 2% to 1%, 5%, 10% or 20%. The result of the above is shown. Good quantification was confirmed in the serum concentration range of 1% to 20%.

[Test Example 2: Calculation of the lower limit of detection of stem cells by the lectin-antibody sandwich method]
Quantitative measurement of podocalyxin was performed using a diluted solution of the culture supernatant of iPS cells (201B7 strain) cultured in different types of media. The following four types of media were used. After culturing iPS cells (201B7 strain) in each medium for 24 hours, the collected culture supernatant was diluted with each medium to obtain a diluted solution.
StemSure hPSC Medium Δ (Wako Pure Chemical Industries, Ltd.)
mTeSR1 medium (STEMCELL Technologies)
MEF-CM (Mouse Embryonic Fibroblast-conditioned Medium, culture supernatant obtained by culturing mouse embryonic fibroblasts)
TeSR-E8 Medium (STEMCELL Technologies)

Biotinylated rBC2LCN lectin (0.3 μg / ml) was added to the wells of the streptavidin plate and immobilized at room temperature (20 ° C. to 25 ° C.) for 1 hour. After washing the wells with a buffer solution (1% Triton X-100, phosphate buffer solution), 50 μl of a diluted solution of the culture supernatant was added to the wells, and the mixture was reacted at room temperature for 1 hour.

After washing the wells with a buffer solution, a peroxidase-labeled R10G antibody (0.5 μg / ml) was added to the wells, and the mixture was reacted at room temperature for 1 hour. After washing the wells with buffer, 50 μl of substrate solution (TMB) was added to the wells and allowed to react at room temperature for 30 minutes. After stopping the reaction by adding 0.5 M sulfuric acid to the wells, the absorbance (450 nm-650 nm) of each well was measured.

The results are shown in FIGS. 4 to 7. In the figure, the horizontal axis indicates the dilution ratio of the culture supernatant, and the dilution ratio of the culture supernatant obtained under the culture condition that the number of cultured cells per 1 ml of the medium is 10,000 is "cultured cells per 1 ml of medium". It is indicated by "equivalent number". That is, "10 x 10 ³ cells / ml" means no dilution, for example, "5.0 x 10 ³ cells / ml" is diluted 2-fold, and "2.5 x 10 ³ cells / ml" is diluted 4-fold. , "1.25 x 10 ³ cells / ml" means 8-fold dilution. In the figure, the linear regression line and the regression equation are the straight line and the equation when the coefficient of determination (R-squared) is the best (close to 1.0).

When any of the four types of media was used, good quantification was obtained even at a high dilution ratio. It was confirmed that the lectin-antibody sandwich method according to the present invention is not affected by the type of medium.

Based on the regression equation, the dilution ratio that is the lower limit of detection was obtained, and the result of calculating the lower limit of detection by "the number of cultured cells per 1 ml of medium" is shown in "Table 1". For the lower limit of detection, the absorbance of the medium alone (the value obtained by adding 3.3 times the standard deviation to the average value of the absorbance) was calculated, and the dilution ratio indicating the absorbance was obtained from the regression equation.

The lower limit of detection, "equivalent number of cultured cells per 1 ml of medium", was 27 to 321 cell / ml. It has been reported that the lower limit of detection in the "lectin-lectin sandwich method" combining rBC2LCN lectin and rABA lectin according to the prior art is 623 to 4,753 cell / ml (see Non-Patent Document 8). It is clear that the "lectin-antibody sandwich method" according to the present invention, which combines rBC2LCN lectin and a hyposulfated keratin antibody R10G antibody, can detect stem cells with higher sensitivity than the conventional "lectin-lectin sandwich method". It became.

[Test Example 3: Detection of stem cells by lectin-antibody sandwich method 2]
In order to evaluate the method for detecting stem cells by the lectin-antibody sandwich method according to the present invention, quantitative measurement of podocalyxin was performed using a diluted solution of the culture supernatant of iPS cells. The measurement was performed by an array detection system using a slide glass.

A non-contact spotter (MicroSystems 4000; Genomic Solutions) was used to immobilize rBC2LCN lectins on epoxy activated glass slides. A diluted solution of the culture supernatant prepared in the same manner as in Test Example 1 was added dropwise to a slide glass on which rBC2LCN lectin was immobilized, and the mixture was reacted at 20 ° C. overnight. After washing, R10G antibody (1 μg / mL) was added dropwise, and the mixture was reacted at 20 ° C. for 1 hour. Next, a Cy3-labeled anti-mouse IgG antibody (Jackson ImmunoResearch, 1 μg / mL) was added dropwise and reacted at 20 ° C. for 1 hour. After washing, it was scanned with an evanescent wave-excited fluorescence scanner (Bio-REX Scan 200, Rexxam).

The results are shown in FIG. In the figure, the horizontal axis indicates the dilution ratio of the culture supernatant as "the number of cultured cells per 1 ml of the medium" as in FIGS. 1 to 4. In the figure, the linear regression line and the regression equation are the straight line and the equation when the coefficient of determination (R-squared) is the best (close to 1.0).

No background due to non-specific binding was observed at any of the dilution ratios, and highly accurate quantitative measurement was possible (coefficient of determination of linear regression line: 0.9997).

From the results of Test Examples 1 to 3, according to the lectin-antibody sandwich method in which rBC2LCN lectin was combined with a hyposulfated keratane antibody R10G antibody, even when the culture supernatant of cells cultured in a serum-containing medium was used. It has been clarified that podocalyxin can be detected with high accuracy and highly sensitive stem cell detection is possible. A similar test was performed using a water-soluble fraction obtained from iPS cells (201B7 strain) cultured in mTeSR1 medium for 24 hours using a commercially available kit (CelLytic MEM Protein Extraction Kit, Sigma-Aldrich). In this case, the same results as when the culture supernatant was used were obtained.

SEQ ID NO: 1: Amino acid sequence of BC2LCN lectin SEQ ID NO: 2: Amino acid sequence of ABA lectin

Claims (12)

A method for detecting stem cells by detecting podocalyxin contained in a cell culture supernatant or lysate.
Culture supernatant or lysate containing serum components and the following (Formula 1):
(R1 represents an OH group or any sugar chain; R2 represents an OH group or any sugar chain, protein, lipid, or other molecule.)
Or the following (Equation 2):
(R1 represents an OH group or any sugar chain; R2 represents an OH group or any sugar chain, protein, lipid, or other molecule.)
The lectin having a binding property to the sugar chain represented by the above is brought into contact with an antibody produced by hybridoma R-10G (deposit number: FERM BP-11301) or an antibody competing with the antibody to obtain the lectin and podocalyxin. The procedure for forming a complex composed of the antibody and
A method comprising a procedure for detecting the complex. The method of claim 1, wherein the antibody comprises Gal-GlcNAc (6S) or a tandem repeat thereof in an epitope. The lectin
A protein comprising the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence in which one or several amino acids of the amino acid sequence are deleted, substituted, inserted, or added, as described in (Formula 1) or The method according to claim 1 or 2 , which is a protein having a binding property to a sugar chain represented by (formula 2). A procedure for contacting the culture supernatant or lysate with the lectin to form a first complex composed of the lectin and the podocalyxin contained in the culture supernatant or lysate.
Any one of claims 1 to 3 , comprising a procedure of contacting the first complex with the antibody to form a second complex composed of the lectin, podocalyxin and the antibody. The method described in. The method of claim 4 , wherein the lectin is bound to an insoluble carrier. The following (Formula 1) contained in the cell culture supernatant or lysate:
(R1 represents an OH group or any sugar chain; R2 represents an OH group or any sugar chain, protein, lipid, or other molecule.)
Or the following (Equation 2):
(R1 represents an OH group or any sugar chain; R2 represents an OH group or any sugar chain, protein, lipid, or other molecule.)
It is a method of detecting stem cells by detecting the sugar chain represented by.
A culture supernatant or lysate containing a serum component, a lectin having a binding property to the sugar chain, an antibody produced by hybridoma R-10G (deposit number: FERM BP-11301), or an antibody competing with the antibody . To form a complex containing the lectin, the sugar chain, and the antibody.
A method comprising a procedure for detecting the complex. The method according to any one of claims 1 to 6 , further comprising a procedure for determining the presence / absence or abundance of stem cells contained in the cells based on the presence / absence or detection amount of the complex. The method according to any one of claims 1 to 6 , further comprising a procedure for determining the differentiation state of the cells based on the presence or absence of the complex or the detected amount. A method for detecting podocalyxin contained in a cell culture supernatant or lysate.
Culture supernatant or lysate containing serum components and the following (Formula 1):
(R1 represents an OH group or any sugar chain; R2 represents an OH group or any sugar chain, protein, lipid, or other molecule.)
Or the following (Equation 2):
(R1 represents an OH group or any sugar chain; R2 represents an OH group or any sugar chain, protein, lipid, or other molecule.)
The lectin having a binding property to the sugar chain represented by the above is brought into contact with an antibody produced by hybridoma R-10G (deposit number: FERM BP-11301) or an antibody competing with the antibody to obtain the lectin and podocalyxin. The procedure for forming a complex composed of the antibody and
A method comprising a procedure for detecting the complex. A kit for detecting stem cells by detecting podocalyxin contained in a cell culture supernatant or lysate containing a serum component .
The following (Equation 1):
(R1 represents an OH group or any sugar chain; R2 represents an OH group or any sugar chain, protein, lipid, or other molecule.)
Or the following (Equation 2):
(R1 represents an OH group or any sugar chain; R2 represents an OH group or any sugar chain, protein, lipid, or other molecule.)
A kit containing a lectin having a binding property to a sugar chain represented by the above, an antibody produced by hybridoma R-10G (deposit number: FERM BP-11301), or an antibody that competes with the antibody . The kit according to claim 10 , wherein the antibody comprises Gal-GlcNAc (6S) or a tandem repeat thereof in an epitope. The kit according to claim 10 or 11 , wherein the lectin is bound to an insoluble carrier.